Process for detecting Borna disease virus (BDV) infections

ABSTRACT

The invention allows, for the first time, to (almost) perfectly detect the various phases of a BDV infection in all species by combining a newly discovered test parameter, the circulating immune complexes (CICs) in the blood plasma, with already known parameters, antigen (AG) in white blood cells and antibodies (AK) in the plasma. All three parameters can be determined from a single blood sample. The detection of BDV-specific CICs described for the first time by the invention as the only infection markers with long-time persistence allows both the detection of healthy carriers (latent infections) and the evaluation of the development of the infection and of therapy results in the case of diseased individuals.

FIELD OF THE INVENTION

The invention relates to a process for detecting BDV infections viadetecting immune complexes which circulate in body fluids (CICs), aprocess for detecting CICs in general, in particular BDV-specific CICs,and to a diagnostic kit which is suitable for these detection processes.

BACKGROUND OF THE INVENTION

BDV infections are found in a large number of livestock and domesticanimals (horses, sheep, cattle, cats) and in humans. BDV is a coatedvirus of 90 nm diameter with a genome of unsegmented single-stranded RNAof negative polarity which encodes 5 genes (genome size: 8.9 kilobases).Related viruses include, for example, the rabies virus and the measlesvirus. Owing to genetic particularities (replication in the nucleus ofthe host cell), BDV is classified as the prototype of a separate familyof viruses (Bornaviridae). BDV has a particular preference for the nervecells in the limbic system of the brain, a functional unit in whichbehavior, emotions and memory functions are controlled. However, othercell types are also attacked by BDV. Peripheral mononuclear leukocytes(PBMCs) are of particular diagnostic importance. BDV does not destroythe host cells (no cytopathogenic effect), neither in the host (in vivo)nor in cell culture (in vitro). The primary pathogenic effect of BDV isbased on a functional disturbance in the infected brain cells which isprobably induced by interaction with neurotransmitter receptors.Experimental data obtained on animals suggest that glutamate receptorsare (reversibly) blocked. The exact mechanism and the receptor type areas yet unknown.

In animals, BDV infections are associated with periodic behavioraldisorders with the indicating signs listlessness, hypoprosexia andataxia. In humans, infectious human Bornaviruses have been isolated frompatients with recurrent endogenous affective disorders, and it is highlylikely that they are involved in these disorders and possibly otherdisorders of the cerebral function (also presumably via neurotransmitterfunctions). Endogenous recurrent depressions, including themanic-depressive form, account for 1-5% of the important psychiatricdiseases and are a considerable health problem owing to the severity ofthe disablement, not only for the sufferers, but, from thesocioeconomical angle, also for society.

BDV infections persist in animals and humans, probably for life. Thesources of the infection remain entirely unknown. The course of theinfection is distinguished by latent and activated phases. During theactivated phases, clinical symptoms can occur. In animals (horses arethe most investigated species), a number of infections without diseaseexist (asymptomatic carriers). This percentage can amount to up to 50%in a group of horses in which an illness was identified. The danger ofan intense, disease-associated activation phase of the infection dependson genetic factors and the stress of the individual (stress factors,immunosuppression).

In humans, there is probably no general risk of illness for healthyinfected individuals which are not predisposed to affective disorders.In contrast, an increased risk of disease by BDV infection in thenarrower sense exists in individuals with a predisposition of developingan affective disorder, in individuals where an affective disorder isalready clinically apparent, and in as yet healthy first-degreerelations of these individuals.

There is an indisputable increasing demand for reliable diagnosticsystems for identifying BDV infection, not only in asymptomatic carriers(for epidemiological reasons), but also for monitoring the infectionphases in diseased individuals.

Conventional antibody tests are known since the BDV virus has alreadybeen studied thoroughly and is already fully sequenced. Until 1993, anantibody test in the serum was the only possibility. As a rule,antibodies are directed against the BDV nucleoprotein p40 (relativemolecular weight 40 kDa) and against the phosphoprotein p24 (24 kDa).These antibodies do not neutralize the virus. It soon emerged that thelack of specific antibodies did not exclude infection with BDV. Thismeans that the antibodies do not persist in the serum of the infectedindividual, or not for a very long time. Moreover, the Ab concentrations(titers) in naturally infected humans and animals are only low in thefirst place and cannot always be identified with less sensitive testsystems.

Important progress, in particular for assessing disease-relevantactivations of the BDV infection, was made by the discovery that PBMCsexpress viral proteins which can be detected in cells after disruptingthe latter. These proteins are occasionally also found in the plasma.Protein (AG) expression has proved to be the decisive activityparameter. It correlates well with clinical disease (humans andanimals), can be quantified and is of great importance for assessing thefurther pathogenesis (chances of recovery, response to antidepressantsand the like). Also, AG determination cannot be replaced by detectingviral nucleic acid in the PBMCs via amplification with nested RT PCR,since it is only the virus itself which is detected by this method, butno information can be obtained on its current or, possibly, futureactivity.

As a rule, the AG marker is limited to part (2-3 weeks) of the acutedisease phase and can no longer be detected during convalescence.Thereafter, the Ab may be detectable intermittently, depending on theseverity of the activation episode, but they may also be completelyabsent.

This current state of BDV diagnostics fails especially duringconvalescence, during the symptom-free interval (which may last foryears) of a patient (humans and animals) and in infected individualswhich are as yet disease-free, i.e., in brief, in the case of BDVinfections in the latent stage. Both test parameters, with AG and Ab,can give erroneous negative results during the latent phase, even thoughthe infection continues to exist.

Journal of Clinical Microbiology, 1997, vol. 35, no. 7, pp. 1661-1666,Horimoto, T. et al. (via MEDLINE TO 97339670) describes an ELISA testfor detecting BDV-specific antibodies in which microwell plates werecoated with BDV p40 antigen in order to catch and detect antibodiesspecific for this BDV nucleoprotein. It was found that many specimenswhich had previously been assayed differently and were found to beseropositive gave a negative result with regard to the antibodies. Itwas concluded that the other studies would have contained a highpercentage of erroneously positive results. However, the results alsoallowed the conclusion that BDV infections cannot be detected in eachindividual case via the antibody titer. Thus, an overall diagnosticapproach seems to be required.

It was therefore the object of the invention to develop a noveldetection process for the infection to allow as complete a detection aspossible of BDV infections, which, owing to their persistence, can lastfor very long periods, with acute and latent phases alternating. Also,it was intended to provide a diagnostic kit which can be used for such adetection.

SUMMARY OF THE INVENTION

To achieve this object, the invention provides a process for detectingBorna disease virus (BDV) infection, in which immune complexes of BDVantigens and specific antibodies attached thereto, which circulatefreely in the body fluid, are detected in a body fluid specimen to betested by means of a suitable immunological assay. An essential aspectof the invention is therefore based on the finding that highconcentrations of circulating immune complexes composed of BDV antigensand antibodies against them which are formed specifically by the bodyoccur in body fluids, for example in blood serum, during certain phasesof the disease. These immune complexes can also be present in particularduring precisely those disease phases during which antigens and/orantibodies cannot be detected.

In principle, the existence of immune complexes, i.e. complexes composedof antigens and antibodies and which are freely mobile in the blood, isalready known, but the relevance of these complexes differs greatly foreach individual case.

For example, NYDEGGER, U. E., in: MASSEYEFF, R. F. [inter alia][Editor].: “Method of Immunological Analysis”, VCH Verlagsgesellschaft,Weinheim [inter alia] 1993, Vol. 1, pp. 646-656 discloses an ELISA assayfor HIV-specific CICs. The authors found high levels of circulatingimmune complexes in HIV-positive drug addicts. They state thatapproximately one tenth of all of the serum IgG present was complexed inCICs. Accordingly, the importance of the CICs is obviouslyvirus-specific. In the case of BDV infections—and in some cases also inHIV infections—the percentage of freely circulating immune complexes canbe very high temporarily, so that most of the “free” antibodies andantigens can be bound therein.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the data of a human patient during hospitalization(treatment with antigen depressants, no antiviral treatment).

FIG. 2 shows a diagram for a 30-year old OCD patient (OCD=obsessivecompulsive disorder) during hospitalization, the diagram correspondingto FIG. 1.

FIG. 3 shows 2 examples from the animal sector, viz. two horsessuffering from Borna disease.

FIG. 4a shows a CSF specimen from a patient with major depressivedisorder.

FIG. 4b shows CSF specimen 137—major depressive disorder, secondepisode, female, 29 years.

FIG. 4c shows another CSF specimen.

FIG. 4d shows CSF specimens from schizophrenia patients.

DETAILED DESCRIPTION OF THE INVENTION

Based on the above finding, a development of the invention introduces anentirely novel diagnostic concept for the virtually completeidentification of persistent BDV infections. The newly discoveredparameter, CIC, is determined in combination with parameters which arealready known, with antigens (AG) and antibodies (Ab), which allows thedetection of various phases of the persistent BDV infection which washitherto impossible. The antigens which are additionally determined inthis context are preferably the BDV nucleoprotein p40 (relativemolecular weight 40 kDa), also termed “N protein”, and the BDVphosphoprotein p24 (relative molecular weight 24 kDa), also termed “Pprotein”. The combined process provides a complete assay system in whichan assay combination composed of the determination of BDV-specificcirculating immune complexes (CICs) in the blood plasma, BDV-specificproteins (antigens, AG) in leukocytes (PBMCs) and in the plasma and/orBDV-specific antibodies (Ab) in the blood plasma is carried out.

The body fluid specimen to be assayed is preferably a blood specimen, itbeing possible for all parameters to be determined on a single bloodspecimen of approx. 10 ml citrated blood.

The blood specimen can be treated as follows:

(a) a blood plasma fraction is isolated from the blood specimen, and thefollowing assays are carried out independently of one another on theblood plasma, spinal fluid or urine specimen:

(1) an antigen detection for BDV antigens,

(2) an antibody detection for BDV-specific antibodies,

(3) a CIC detection;

(b) a blood plasma fraction and a leukocyte fraction are prepared fromthe blood specimen, and the assay in accordance with (a) (1) is carriedout on the leukocyte fraction—in the case of a blood specimen—and/or onthe blood plasma fraction.

By means of the invention, it was possible for the first time todemonstrate with reference to a large number of assays that the CICsplay a key role in diagnostics, but also in the understanding of thecourse of the infection. As has been found, CICs persist considerablylonger than soluble BDV antibodies which can be identified with Abassays and longer than cell BDV AG, which is only found during the acuteclinical phase. CICs can still be detected after prolonged symptom-freephases when Ab assay and AG assay are negative, and allow the conclusionthat activation phases during which AG was liberated have taken placeand that Ab which complex this AG have existed.

By introducing the CICs as diagnostic assay parameter which is used, ifappropriate, in combination with Ab and AG, the invention closes adiagnostic gap which has previously been impossible to close.

It is known for a variety of systems that circulating immune complexes,i.e. freely mobile AG/Ab complexes, exist in principle. Free complexformation of AGs with Ab in the serum and vice versa has already beenexploited for a variety of immunological assays. The existence of CICsin BDV infections, in particular the long-term high CIC concentration,even during the latent interval, and the associated diagnostic relevanceof the CICs, was hitherto completely unknown.

In some patients with endogenous affective disorders and BDV infections,weekly controls even during the acute phase show that extremely high CICconcentrations, but no free Ab and no AG can be measured in PBMCs.Conventional diagnostics would miss these patients completely. With sucha course of the infection, it must be assumed that the BDV-AG formed inthese PBMCs in high concentrations immediately enters the plasma and isbound by the Ab present in CICs.

This is why the CIC determination described in the invention, inparticular the assay combination of CIC, AG and Ab measurement, meetsfor the first time requirements of persistent BDV infection with itscourse, which varies between latency and activation, and has thereforegained great importance, not only for the diagnostic monitoring ofdiseased individuals, but also for identifying latent infections inhealthy individuals.

In the case of high-risk patients, an obligatory CIC determinationpermits a BDV diagnostic which is no longer limited to the acuteillness. While owing to the low viral activity the study of latentinfections in healthy individuals was possible theoretically, albeitonly with frequent checks, this was virtually impossible under realisticconditions. The use of the CIC assay and the assay combination allowlatent infections of healthy individuals to be detected, which is ofepidemiological importance since the infection sources and thetransmission mechanisms remain unknown in humans and animals.

The use of the processes according to the invention is suitable foridentifying BDV infections in humans and animals and possibleindependently of the clinical illness. The detection process accordingto the invention allows the quantitative measurement of BDV-specificCICs for humans and various animal species.

The term “detection” is always used in the terminology of the presentinvention in such a way that both a first, qualitative detection and aquantitative measurement of the relevant CIC concentrations in the formof a titer are possible.

The invention proposes as suitable immunological assay or suitableprocess for detecting immune complexes which circulate in body fluids aprocess with the following steps:

(1) fixing, via the Fc region in a suitable manner on a carrier whichmay have been prepared for this purpose, monoclonal or polyclonalantibodies which specifically bind to the antigens contained in theCICs;

(2) bringing into contact with the antibodies a body fluid specimen,preferably a blood plasma specimen, which is to be assayed for thepresence of CICs;

(3) bringing into contact with the specimen treated in accordance withsteps (1) and (2) a secondary antibody of a species other than theassayed species, preferably a goat-anti-species antibody, which isspecific for antibodies of the species whose body fluid specimen wasused;

(4) detection and/or measurement of the quantity of the secondaryantibody by a suitable immunological detection process.

For this purpose, the antibodies used in step (1) are preferablymonoclonal BDV-specific N and/or P protein antibodies which have beenobtained, for example, from mice.

The support which may have been prepared in a suitable manner can be anabsorptively fixing polymer assay plate or a corresponding test tube,this plate or the tube preferably first being occupied as completely aspossible with secondary antibodies which are directed specifically forthe species from which the CIC-antigen-specific antibodies wereobtained, and, in a subsequent plate preparation step, theCIC-antigen-specific antibodies being applied to this layer of secondaryantibodies.

Alternatively, fixing of the CIC-antigen-specific antibodies in step (1)of the process may also be effected with a suitable, different process,which allows the antibodies to be fixed securely on the plate in thesuitable orientation, for example by applying a Clq base layer to apolystyrene support. The application of such a base layer generallypermits a more economical use of the more expensive CIC-specificantibodies.

The detection in accordance with step (4) of the process can beeffected, for example, via an enzyme-coupled secondary antibody at whicha color reaction is triggered with a suitable substrate. In thecurrently preferred embodiment, the secondary antibody is coupled toalkaline phosphatase and visualized, or made measurable by means ofoptical detectors, with p-nitrophenyl phosphate by exploiting thereaction between the alkaline phosphatase and the para-nitrophenylphosphate, which leads to a yellow color.

A secondary antibody for the purposes of the invention is an antibodywhich is not specific to an antigen, but to another antibody, i.e. anantibody, of a different species, which is recognized as “foreign”.

CIC determination can be effected in any body fluid which contains theseCICs in sufficient concentration. In the currently preferred embodiment,the body fluid preferably used for the assay is a blood plasma specimenobtained from citrated blood. However, spinal fluid may also be used,inter alia.

BDV antigens and BDV-specific antibodies are required for the assaycombination. The antigen source may be BDV-infected cells. The assay isnot influenced or distorted when (homogenized) antigen-containing cellsuspensions are used directly instead of isolated antigens. In an assaycited in the examples (see “Ab assay”), for example, a dilution of a 10%brain suspension of a horse which had died from Borna disease was used.

The applicant has deposited, at the Deutsche Sammlung fürMikroorganismen und Zellkulturen [German Collection of Microorganismsand Cell Cultures], fetal human oligodendroglia cells which grow as acell line, are persistently infected with BDV, and are free from otherviruses and from mycoplasmas. This deposition was done in accordancewith the provisions of the Budapest Treaty on Dec. 12, 1997; the nameand identification number “DSM ACC 2334” was assigned to the cultureOLIGO/TL.

The cell line originates from the applicant's material. The fetal humanOL cells are persistently infected with BDV, without CPE. The internalname of this cell line, which has been passaged approximately 110 times,is OLIGO/TL (or OL/TL).

Antigen which is suitable for the use in accordance with the inventioncan be obtained at any time from these cells. Also, other cell lines,for example also animal cell lines, can be infected with the aid of thiscell line. The titer is approx. 10³ FFU/ml. In particular, the cellsalso contain the above-described antigens p40 and p24.

BDV-specific monoclonal or polyclonal antibodies can be produced at anytime in a manner known per se with the aid of the cell suspension assuch or else with proteins isolated therefrom. Monoclonal N and Pantibodies from mice are currently preferred. To this end, mice areinfected or immunized, and hybridoma cultures produced from their Bcells, as usual. The supernatants are assayed (screened) for antibodieswhich “stain” BDV-specifically. For obtaining antibodies, see also “H.Ludwig et al., Arch. Virol. (1993) Suppl. 7:111-113”.

The invention furthermore encompasses a diagnostic kit for detecting BDVinfections. This kit, or this assay or detection system, comprisesBDV-specific monoclonal or polyclonal antibodies, means for contactingthese antibodies with a specimen suspected of containing BDV antigens orBDV CICs, and means for detecting the attached antigens orantigen/antibody complexes (CICs). Alternatively, the kit containsBDV-specific monoclonal or polyclonal antibodies occupied with BDVantigens, means for contacting these antigen-occupied antibodies with aspecimen if the latter is suspected of containing BDV antibodies, andmeans for detecting the BDV antibodies which attached themselves in thiscase.

The diagnostic kit can further encompass a unit on which, or in which,the BDV-specific antibodies are present in immobilized form. Such a unitcan be an assay plate which has been prepared in a particular manner, ora test tube which has been coated on the inside.

In a preferred immobilization method, the BDV-specific antibodies aremonoclonal or polyclonal antibodies obtained from a species I which havebeen immobilized by being applied, and thus held in place, on a support,preferably in the form of an assay plate or a test tube, which has beencoated with a species II-anti-species I IgG obtained from a differentspecies (species II).

Also preferably, the antibodies of species I are polyclonal ormonoclonal mouse antibodies, preferably monoclonal mouse antibodieswhich are specific for protein P and/or protein N, the adsorptivecoating of the support being composed of an anti-mouse-IgG, preferably agoat-anti-mouse-IgG. Alternatively, the BDV-specific antibodies can alsobe fixed on or in the unit via polystyrene-bound Clq, or immobilized byany other suitable method.

EXAMPLES

To facilitate the understanding of the invention, the technical detailsof the assay combination from the treatment of the blood specimen to theevaluation are described hereinbelow. The application is illustrated byindividual examples. The examples are exclusively intended forillustrative purposes. Naturally, the principles of the invention canalso be realized in a different fashion.

Part I: Processing of the Specimen Material to be Assayed

10 ml of citrated blood are required for the test for BDV infection. 9ml of venous volunteer blood are added to 1 ml of an 0.106-molar sodiumcitrate 2-hydrate solution and the specimen is mixed thoroughly. It isrecommended to use ready-to-use citrate tubes, for example the 10 mlMonovette 9NC by Sarstedt. Until the specimen is sent to the testlaboratory, it should be stored at 4° C. The specimen quality is notadversely effected when posted by express delivery without refrigeration(1 to not more than 3 days).

The citrated-blood specimen is separated using density-gradientcentrifugation into the plasma fraction and the cellular bloodcomponents. The plasma fraction is used to assay for CIC, AG and Ab. Thecell fraction of the leukocytes (PBMCs) is processed and then used forAG assaying. If the intention is only to assay for CICs, or if onlyplasma assays are carried out, the cell fraction can be discarded.

To separate the components, Ficoll separating solutions (Biochrom) ofdifferent densities are used, depending on the animal species.

The following are required for 6 ml of blood:

Humans 3 ml Ficoll, density 1.077

Horses 3 ml Ficoll, density 1.090

Cattle 2 ml Ficoll, density 1.077+1 ml Ficoll, density 1.068

Cats 2.8 ml Ficoll, density 1.077+0.2 ml PBS pH 7.2

Dogs 2.8 ml Ficoll, density 1.077+0.2 ml PBS pH 7.2

Rabbits 2.8 ml Ficoll, density 1.077+0.2 ml PBS ph 7.2

I. Protocol for Separating a 10 ml Citrated-blood Specimen

(1) Fill conical 10 ml disposable tubes with 3 ml Ficoll separatingsolution of the relevant density and superimpose a layer of not morethan 6 ml citrated blood

(2) Centrifuge specimen for 20 minutes at 1049 g (2200 rpm, HeraeusChrist Minifuge) (in the case of all specimens with Ficoll of density<1.090); in the case of Ficoll of density of 1.090, centrifuge for 20minutes at 1249 g (2400 rpm)

(3) Carefully remove plasma fraction (supernatant), using disposablePasteur pipette; for immediate assaying the same day, store at 4° C.;for later assaying, store at −20° C.

(4) The leukocyte fraction forms a discernible ring on or above theFicoll, while the erythrocyte fraction (not required) is present in theFicoll as a pellet. After removal of the plasma, remove cell ring usingdisposable Pasteur pipette and transfer into a fresh conical 10 mlplastic tube, make up with PBS and mix thoroughly

(5) Centrifuge leukocytes (PBMCs) for 10 minutes at 1952 g (3000 rpm).Discard supernatant and take up pellet in not more than 0.5 mlPBS=approx. 10-fold concentration. Transfer solution into sterilefreezer tubes (for example Nunc, 1.5 ml) equipped with screw top.

(6) For immediate assaying of internal BDV antigens (see below), storethe cells in PBS at 4° C.; for assaying at a later time, store at leastat −20° C., better at −70° C.

II. Assay Systems

An assay combination of EIA (enzyme immunoassay)—basis (solid-phaseassay) is used for assaying AG in PBMCs and/or in blood plasma and CICand Ab in plasma. The first two assay steps are the same for all threeassay systems. The subsequent description in the form of assay protocolsis therefore divided into (A) coating of the assay plates (A1) and (A2)and (B, C, D) protocol for the AG, CIC and Ab assay.

EIA Protocol (Double-sandwich Type)

(A) Protocol for Coating the EIA Plates

Maxi Sorp immunomodules (Nunc® Maxi Sorp F8, Cat. No. 469949) were usedas polymer supports for the subsequent reactions. One frame accommodates12 modules with 8 vertically arranged shallow polymer recesses and haveexactly the dimensions of a 96-well microtiter plate (standard dimensionfor EIA systems):

(A1) 0.1 ml per well AffiniPure® goat-anti-mouse IgG, Fc fragment,absorbs against human, bovine, equine serum proteins (Dianova® Cat. No.115-005-071), diluted 1:1000 (=1.8 micrograms protein per milliliter) incoupling buffer; incubation overnight at 4° C. or for 2 hours at 37°.

Wash 3× in wash buffer (Dynatech 96-well plate washer)

(A2) 0.1 ml per well monoclonal antibodies against the BDV N-protein p40and BDV P-protein p24 (W1 and KFU2, ref.: Arch. Virol. (1993) Suppl.7:111-133) (as catch antibodies), in each case diluted 1:500 in dilutionbuffer. Incubation for 2 hours at 37° C. or overnight at 4° C.

The plates can then be stored at 4° C. up to at least 4 weeks beforebeing used further.

(B) Protocol for the Determination of BDV Antigens (AG) in PBMCs

Preparation

Wash the A2-coated plates 3× in wash buffer

Ultrasonic treatment (20 cycles min⁻¹, 40 mA) (Branson Sonifier B15P;ref. Mol. Psychiatry (1996) 1:200-212) of the PBMCs isolated in I.

AG Assay

(B1) Specimen dilution: into each well of the precoated plates introduce0.1 ml dilution buffer, add in A sonified PBMCs (0.1 ml) or blood plasma(0.1 ml) (=1:2) and continue diluting a further 7 steps to basis 2 (to1:256); incubate overnight at 4° C.

Wash 3× in wash buffer

(B2) per well 0.1 ml BDV-specific rabbit antiserum (for example GC 12,immunofluorescence titer 1:1000) (=detection antibody) in suitabledilution (1:1000, 10-fold more concentrated than the IF titer) indilution buffer; incubate for 2 hours at 37° C.

Wash 3× in wash buffer

(B3) per well 0.1 ml alkaline-phosphatase-coupled Affini Pure®goat-anti-rabbit IgG, Fc-fragment-specific, absorbs against human serumproteins (Dianova®, Cat. No. 111-055-046) (=secondary antibody), diluted1:3000 in conjugate dilution buffer; incubate for 1 hour at 37° C.

Wash 3× in wash buffer

(B4) per well 0.1 ml of the substrate solution (1 mg/ml p-nitrophenylphosphate; Sigma, obtainable as substrate tablets) in substrate dilutionbuffer; incubate for 5-10 minutes at room temperature or until the colorreaction develops (color change from colorless to yellow) in thepositive control.

(B5) per well, add 0.05 ml of stop solution (3-molar NaOH solution)

(B6) Measure at 405 nm in a microtiter plate Multiscan (for example BIORAD 2550 EIA READER or Dynatech); blank measurement against unreactedsubstrate. Semiquantitative evaluation sufficient: (+)=weekly positive,to a maximum of 4+=extremely positive; +/−means inconclusive andrequires a second test. (Example see under Use).

(C) Protocol for the Determination of BDV-specific Circulating ImmuneComplexes (CICs) in the Blood Plasma

Preparation

Wash the A2-coated plates 3× in wash buffer

CIC Assay

(C1) Specimen dilution: into each well of the precoated plates,introduce 0.1 ml dilution buffer; in A: add 0.09 ml dilution buffer+0.01ml of the blood plasma prepared under I. (=dilution 1:20), continuediluting a further 7 steps to basis 2 (to 1:2560]; incubate for 1-2hours at 37° C. (standard time: 1 hour at 37° C.).

Wash 3× in wash buffer

(C2) per well 0.1 ml alkaline-phosphatase-coupled goat-anti-species(corresponding to the species of the specimen) IgG, Fc fragment (alsoobtainable from Dianova®) (=secondary antibody), dilution 1:3000 inconjugate dilution buffer; incubation for 1 hour at 37° C.

Wash 3× in wash buffer

(C3) per well 0.1 ml of the substrate solution (1 mg/ml p-nitrophenylphosphate; Sigma, obtainable as substrate tablets) in substrate dilutionbuffer; incubate for 5-10 minutes at room temperature or until the colorreaction develops (color change from colorless to yellow) in thepositive control.

(C4) per well, add 0.05 ml of the stop solution (3-molar NaOH solution).

(C5) Measure at 405 nm in a microtiter plate Multiscan (for example BIORAD or Dynatech); blank measurement against unreacted substrate.Semiquantitative evaluation from (+) to 4+ sufficient (cf. also AssayB); however, titer indications with end-point determination alsopossible. (Examples see Use).

(D) Protocol for the Determination of BDV-specific Antibodies (Ab) inthe Blood Plasma

Preparation

Wash the plates coated up to A2 3× in wash buffer

Ab Assay

(D1) per well 0.1 ml of a 10% brain suspension (=detection antigen) of ahorse which has died from Borna disease, in a suitable dilution (1:50)in dilution buffer; alternatively, use can be made of cell culturesupernatant of the cell line deposited on 12.12.1997 under No. DSMACC2334 at the DSMZ which is persistently infected with a Borna diseaseviral strain, diluted between 1:100 and 1:300 in dilution buffer,

[lacuna] of blood plasma, prediluted 1:5 (=dilution 1:50), predilute afurther 7 steps to basis 2 (to 1:6400); incubate for 1 hour at 37° C.

Wash 3× with wash buffer

(D3) per well 0.1 ml of alkaline-phosphatase-coupled goat-anti-species(corresponding to the species of the specimen) IgG, Fc-fragment-specific(Dianova®) (=secondary antibody), diluted 1:3000 in conjugate dilutionbuffer; incubate for 1 hour at 37° C.

Wash 3× in wash buffer

(D4) per well 0.1 ml of the substrate solution (1 mg/ml p-nitrophenylphosphate; Sigma, obtainable as substrate tablets) in substrate dilutionbuffer; incubate for 5-10 minutes at room temperature or until the colorreaction develops (color change from colorless to yellow) in thepositive control.

(D5) per well, add 0.05 ml of the stop solution (3-molar NaOH solution).

(D6) Measure at 405 nm in a microtiter plate Multiscan (for example BIORAD or Dynatech); blank measurement against unreacted substrate.Evaluation possibilities as in Assay C.

III. Buffer Solutions

Coupling Buffer

Stock solution A: 0.02 M NaH₂PO₄.H₂O 2.76 g; dd—water to 1000 ml

Stock solution B: 0.02 M Na₂HPO₄.2H₂O 3.561 g; dd—water to 1000 ml

Working Solution

65 ml solution A+45 ml solution B+14.6 g NaCl, dd—water to 1000 ml, pH7.6.

Wash buffer: NaCl 45 g   (9 g per 1000 ml) Tween 20 2.5 g (0.5 g per1000 ml) NaN₃ 1.0 g (0.1 g per 1000 ml) dd - water to 5000 ml

Dilution buffer: KH₂PO₄ 0.2 g Na₂HPO₄ · 12H₂O 2.9 g NaCl 8.0 g KCl 0.2 gTween 20 0.5 g NaN₃ 0.2 g dd - water to 1000 ml, pH 7.2 must be checkedor established.

Conjugate dilution buffer (TBS-TWEEN): Dissolve in 900 ml dd - waterTRIS 2.4 g NaCl 8.0 g KCl 0.2 g Tween 20  0.5 g,

bring to pH 8.0 with concentrated HCL, then make up to 1000 ml.

Substrate dilution buffer (DIETHANOLAMINE BUFFER): Dissolve in 843 mldd - water diethanolamine 97 ml MgCl₂ 0.1 g NaN₃ 0.2 g, then add 60 ml 1N HCL, bring to pH 9.8.

Stop solution (3 M NaOH): NaOH 120 g dd - water to 1000 ml

IV. Sources of Commercially Available Reagents for the Assay SystemsDescribed

1. Ficoll separating media: Biochrom KG, D-12247 Berlin

2. Citrate tubes: Sarstedt, D-51588 Nümbrecht

3. EIA microtiter strips (Maxi Sorp immunomodule) F-form: Nunc,Roskilde, Denmark

4. Enzyme conjugates for secondary antibodies: Dianova, manufacturer:Jackson Immuno Research Labs Inc., West Grove, Pa., USA

5. Washer for EIA assays: Dynatech Ultrawash plus; Dynatech Labs. Inc.Chantilly, Va., USA

6. Substrate for enzyme conjugates (for alkaline phosphatase): SigmaChemical Co., St. Louis, Mo., USA

7. Buffer substances (in III) with the exception of KCL: Merck KGaA,D-64271 Darmstadt, Fluka Chemie AG, CH-9471 Buchs

8. Photometer (for microtiter plates): BIO RAD Labs. Hercules, Calif.,USA or Dynatech Labs Ind., Chantilly, Va., USA

9. Sonifier for cell disruption of the PBMCs: Branson Sonifier, B15JP,Branson Sonic Power Company, Danbury, Conn., USA

V. BDV-specific Reagents

1. Monoclonal Antibodies: Needed as Base Coating (Step A2) for Assays B,C, D

1a) W1, recognizes a sequential epitope on the N-protein (p40) of BDV.The binding site is conserved, i.e. is recognized by more than only thehost species. The antibody is characterized in the reference Arch.Virol. (Suppl.) (1993) 7:111-133.

1b) KFU 2, recognizes a sequential epitope on the P-protein (p24) ofBDV. The binding site is conserved, i.e. is recognized by more than onlythe host species. The antibody is characterized in the reference Arch.Virol. (Suppl.) 7:111-133 (1993).

2. Rabbit Antisera (Specific Detection Antibodies): Required for Assay B(AG) in Step B2

It is possible to use the sera of experimentally infected rabbits(reference as under V, 1a). They must have a minimum titer of 1:2000 inthe immunofluorescence assay and recognize at least the N- andP-proteins of BDV (p40 and p24) in the Western blot. Since Borna virusesare genetically highly stable according to present knowledge, i.e. thesequences in different viral strains show only minor differences,polyclonal antisera generally recognize the proteins of Borna virusesfrom different species.

This also applies to the few human strains which have been obtained todate (reference Mol. Psychiatry (1996) 1:200-212; Virus Res. (1996)44:33-44.), even though the human strains show relevant point mutationsat amino acid level which have not been found as yet in animal strains.

In addition to the serum GC12 which has been mentioned in thedescription of the assay and other antisera obtained by infection withthe animal reference strain V, it is possible to use defined rabbit serawhich have been generated by experimental infection with the differenthuman BDV strains (reference Mol. Psychiatry (1996) 1:200-212) and whichrecognize the entire spectrum of viral proteins.

3. Defined Antigen Suspensions Required for Assay D (Ab Assay) in Step D

Brain suspensions (10%) of horses which have died from Borna diseasecontain a high concentration of viral proteins (in particular N- andP-proteins). A progressive neurological disease which develops as aconsequence of BDV infection and ends in death is rare compared withperiodically occurring, spontaneously remittent behavioral disorders orBDV infections with a subclinical course (reference: Arch. Virol (1997)Suppl. 13:167-182). The encephalitis which is termed the so-calledclassic Borna disease is probably caused by a virus production which hasgone out of control. The virus titers in the brain reach 10⁵ to 10⁶FFU/ml and are thus within the range of experimentally infectedlaboratory animals (reference: Prog. med. Virol. (1988) 35:107-151).

Antigen suspensions which can be employed are directly defined brainsuspensions whose virus and antigen titers have been determined.Suspensions from cell lines which are persistently infected with BDV(human and animal strains) can also be used for the Ab assay asdescribed. These persistently BDV-infected cell lines are particularlysuitable for standardization purposes. Cell lines which can be used aredeposited at the DSMZ in accordance with the provisions of the BudapestTreaty under number DSM ACC 2334 (date of deposition Dec. 12, 1997).

Defined antigen solutions can be employed as positive control in assay B(AG assay).

VI. Brief Description of the Test Principles of the Assay CombinationDescribed in the Invention

The assay combination is designed as a triple assay for three differentBDV test parameters which are relevant for the infection, viz. Antigen(AG, assay B), circulating immune complexes (CIC, assay C) andantibodies (Ab, assay D) in a single blood specimen. The assays can alsobe carried out individually, for example for testing very specificputative phases of the disease. The assays are designed as solid-phaseassays and are evaluated photometrically. Assay reactions and procedurestake place in microtiter systems, which are modern laboratory standards.

Part A, which is used as base coating, is exploited equally by allthree, or four, assays. The base coating is a core piece of the assaycombination and is responsible for the high BDV specificity. The latteris generated by a set composed of two monoclonal antibodies which aredirected against conserved epitopes of the N- and P-proteins of Bornavirus (p40, p24). A very efficient use of these catch antibodies isachieved by a precoating with an anti-mouse antibody. Without thisprecoating, it is necessary to subject the specific monoclonalantibodies to purification by affinity chromatography. In assay B forthe AG detection, p40 and p24 proteins which are (freely) present in theblood plasma or in the PBMCs are bound by the catch antibodies.Polyclonal rabbit detection antibodies, which are also BDV-specific,recognize the bound antigen from the specimen and it is visualized bythe enzyme-coupled indicator system. This double-sandwich principle hasthe advantage that small amounts of BDV antigen become detectable in alarge quantity of cell proteins owing to the specific binding to twodifferent antibody systems.

In assay C for the CIC detection, the antigen moiety of BDV immunecomplexes present in the plasma is bound by the catch antibodies. In thenext reaction step, the antibody moiety of the immune complex is boundby an enzyme-labeled anti-antibody (secondary antibody), and thisbinding is visualized via the indicator system. This assay design, whichis amazingly simple under realistic conditions, visualizes only thoseCICs which are BDV-specific and thus diagnostically relevant. This assayis the core of the invention and closes the diagnostic gap which hasexisted to date. BDV-specific CICs were discovered by the inventors, andtheir detection was developed.

In test D for the Ab detection, BDV antibodies to p40 and p24 which are(freely) present in the plasma are determined via a detection antigensolution which has previously been bound by the catch antibodies. Thespecifically bound antibodies are visualized via enzyme-labeledanti-antibodies (secondary antibodies), the indicator system. The assaydesign has the advantage that unpurified infected brain or cell culturesuspensions can be used as detection antigen solution without a loss inspecificity. The specificity is guaranteed by the catch antibodies.

A purified antigen solution will always be required for other, moresimple EIA-based Ab assays, and this purified antigen solution must bebound to the polymer support as a solid phase. While these assays aremore rapid to carry out, antigen purification means that they arecomplicated and expensive.

The standard BDV antibody assay currently carried out in otherinstitutions is the indirect immunofluorescence assay (reference: J.Med. Virol. (1992) 36:309-315). The assay has the advantage over newsystems in that the results can be compared quantitatively andqualitatively with those from other laboratories (Review: CTMI (1995)190:103-130). Immunofluorescence titers are generally lower than EIAtiters. In the longitudinal-section studies shown in Section VII, theantibodies were detected by immunofluorescence to ensure that they canbe compared with other laboratories'results obtained on a merelyserological basis.

The EIA-based Ab assay shown in the assay combination improves thedetectability of the antibodies owing to its higher sensitivity, butultimately changes nothing regarding the drop below the detection limitand below titers described in the introduction. This is why a lack of Abdetection with conventional methods does not exclude BDV infection. Theoccurrence of free antibodies, which is only a temporary phenomenon, canbe explained by the formation of CICs.

The four assay parameters define the following diagnostic principle:

cAG: The antigen assay from PBMCs is suitable as acute marker for virusreplication. cAG can only be measured briefly during the disease phase.

pAG: The antigen expressed into the plasma is the result of virusreplication in the cells. It generally appears somewhat later than cAG,but sometimes also simultaneously, and can frequently be detected overseveral weeks. pAG is also an acute marker during the disease phase.However, its detectability depends on the rate at which the antibodiesand subsequently the CICs are formed. Course studies (weekly) have foundthat only pAG is measured positively in some specimens (for example week1; only pAG, week 2: pAG+CIC etc.).

CIC: The circulating immune complexes are very good infection markersand therapy control markers. With antiviral therapy, disappearance ofthe CICs, or no neogenesis, are observed over a prolonged period. Thehalf-life of the CICs is four weeks in theory; however, CICs canfrequently still be detected many weeks after the acute pathologicalprocess. No therapy means that an alternating CIC level is generallyretained. CIC measurement is therefore also suitable for healthyindividuals as screening for latent infections.

Ab: Antibodies only occur when stimulated by the antigens. During theacute phase, the antibody titer is low or not detectable since theantibodies are bound in the immune complexes. The antibodies can bedetected better during convalescence. (Note: it must be noted that thedata published earlier by third parties always refer to theimmunofluorescence assay (IF), which is considerably less sensitive, sothat antibodies could only be detected occasionally.

In the use examples which follow, it is demonstrated that the stage ofthe infection changes constantly (especially during the disease phase)and that the detection of a plurality of parameters is idealdiagnostically for this type of a persistent virus infection (with a lowreplication rate during the activation phases).

VII. Use Examples of the Invention

Three case examples from the human sector and two case examples from theanimal sector are described in the following text. These examples wereselected from a large number since it was possible to study anabove-average specimen size (up to 23 from one individual) overespecially long observation times of up to 7 months.

Human Patient 1: (Initials D. R.)

Sex: Male

Age: 59 years

Clinical diagnosis: recurrent endogenous depression (rMDD-recurrentmajor depressive disorder), plus neurotic aspects

Examination: during the acute depressive episode

Observation time: 27 weeks (approx. 7 months, May-November 1996)

Number of specimens: 23, of which 13 at 1-week intervals

Summary of the assay result:

Antigen: 4/23

CIC: 23/23

Antibodies: 0/23

Human Patient 2: (Initials G. R.)

Sex: Female

Age: 76 years

Clinical diagnosis: rMDD

Examination: during the acute depressive phase

Observation time: 21 weeks (approx. 5 months, June to November 1996)

Number of specimens: 18 (of which 14 in 1-week intervals)

Summary of the assay result:

Antigen: 4/18

CIC: 17/18

Antibodies: 3/18

Human Patient 3: (Initials A. J.)

Sex: Male

Age: 63 years

Clinical diagnosis: rMDD

Examination: during the acute depressive episode

Observation time: 6 weeks (August to September 1996)

Number of specimens: 7, all at 1-week intervals

Summary of the result:

Antigen: 1/7

CIC: 4/7 (2 specimens +/−)

Antibodies: 1/7

Equine Patient 1: (A. S., from North-Rhine Westphalia)

Sex: Male, gelding, Hannoverian

Age: 11 years

Clinical diagnosis: Borna disease, severe episode of apathy, somnolence,loss of appetite, altered feeding behavior; spontaneous remission (ArchVirol (1993) Suppl 13, in press)

Examination: during the acute disease phase and after convalescenceStart: 3 weeks after the beginning of the illness)

Observation time: 28 weeks (7 months, June 1995 to January 1996)

Number of specimens: 8 (6 complete examinations)

Summary of the assay result:

Antigen: 4/6

CIC: 4/6

Antibodies: 5/6

Equine Patient 2: (F. D.) from Bavaria

Sex: Male, Bavarian gelding

Age: 15 years

Clinical diagnosis: Borna disease, severe neurological form, euthanasia(August 1996); first episode in 1994, with spontaneous remission

Observation time: 14 weeks (May to August 1996)

Number of specimens: 3 blood specimens, 1 spinal fluid specimen

Summary of the assay result:

Blood:

Antigen: 2/3

CIC: 3/3

Antibodies: 0/3

Spinal fluid:

Antigen: Finding +/0

CIC: 1/1

Antibodies: 0/1

The age distribution of the human patients in the case examples selectedis atypical. There are similar courses from all age groups. The coursesare typical for affective diseases with recurrence (either rMDD orbipolar illness), examined during the acute disease phase.

During the symptom-free interval, low CIC concentrations usually remain,while antigen in PBMCs and antibodies are no longer detectable. Theduration of CIC persistence, and the titer level, depend on theintensity of the previous activation phase of the BDV infection.According to the examinations which we have carried out so far, the CICspersist for at least 3 months (after the disease episode). No systemicdata exist for very long symptom-free intervals (for example severalyears). We know from individual examinations that even the CICs candisappear. When a new depressive episode starts after a long healthyinterval, the status during the initial examination can be negative forall 3 parameters. To guarantee that BDV infection can be excluded, asingle examination is therefore not sufficient. Rather, at least onesecond examination during the first third of the disease episode isnecessary. If infection is present, then at least CICs are found, as arule, while the AG or Ab findings are still negative.

Under realistic conditions, this means that, taking into consideration atime interval, BDV infection can be detected sufficiently reliably bythe CICs shown in the invention and by the novel triple test which alsotakes into consideration AG and Ab.

The case examples of the equine patients deal with diseased, infectedanimals. Healthy, symptom-free carriers of the infection can frequentlyonly be detected by CICs. Examples of examinations in healthy groups areshown under X.

Comment

Human patient 1 is a specific case since he belongs to the small numberof patients where almost exclusively exorbitantly high CICconcentrations, but no free antibodies and only rarely AG in PBMCs weredetectable in weekly checks during the acute disease phase (see alsointroductory section). We assume that the BDV-AG formed in the PBMCsimmediately enters the plasma and is bound in CICs by the antibodieswhich are present. Conventional assay methods which do not take intoconsideration the CICs would virtually not identify patients of thistype.

Possibilities of Evaluating AG and CIC assays

As a rule, a semiquantitative evaluation which indicates the relativeconcentration of the parameters is preferable to a titer value. Eachparameter is measured in an 8-step dilution series per specimen. Theabsorption, which drops in parallel with the dilution, provides anadditional reliability when assessing the titer. It can be used for anend-point titer determination, but does not need to be.

VIII. Using the Assay Combination for Assessing the Actual State of theBDV Infection

The AG detection in PBMCs (and in the blood plasma) is a parameter foran acute activation process. The CIC parameter assesses the pastinterval during course studies. When CIC values rise within theinterval, this means that viral antigen has been formed and alreadyexists in complexed form at the time of assaying. Whether freeantibodies are detectable is determined by the antibody production inrelation to the antigen production. If antibody production has only justrestarted owing to an activation episode, free antibodies will hardly bedetectable, but CICs will. At a later point in time, when antigenproduction is down, the chance of detecting free antibodies increases(with or without simultaneous CIC detection).

An interpretation of the triple test which takes into considerationthese circumstances is very helpful for assessing clinical progresses.

The following assay constellations are possible, according to thepresent-day art:

1. AG-negative CIC-negative Ab-positive

Infection is present, but has not been activated, neither acutely norduring the last weeks.

2. AG-positive CIC-negative Ab-negative

Infection is highly acutely activated at the time of examination, butnot before.

[Lacuna]

Infection is present, activation started a few weeks ago, but activationis still acute at the time of examination.

4. AG-negative CIC-positive Ab-negative or -positive

Infection is present, but activation began a few weeks, possibly monthsago (depending on the examination interval). Activation no longerpresent at the time of examination.

If this is an initial examination, only the statement “infection ispresent” can be made.

5. AG-negative CIC-negative Ab-negative

No suggestion that BDV infection is present. If two assays are fullynegative during the acute disease phase, infection can be excluded.

To monitor the therapy before terminating an antiviral treatment, atleast two assays (interval 4-6 weeks) must be completely negative(Lancet (1997) 349:178-179).

Earlier studies have shown that CICs are a highly sensitive marker forassessing the success of antiviral therapy. They can frequently still bedetected as the last parameter after clinical remissions have alreadytaken place, but disappear later when the therapy is continued.

IX. Detection of Infection in Symptom-free Horse Populations, Deleted

The invention allows for the first time the various phases of a BDVinfection to be identified (virtually) completely in more than onespecies by combining the newly-discovered assay parameter “circulatingimmune complexes”, (CICs) in the blood plasma, with parameters which arealready known, viz. antigen (AG) in leukocytes and antibodies (Ab) inthe plasma. All three parameters can be determined on a single bloodspecimen. The detection, which is described for the first time in theinvention, of BDV-specific CICs as the only long-term persistentinfection markers allows not only healthy carriers (latent infections)to be identified, but also the course of the infection and the successof the therapy to be assessed in the case of diseased individuals.Immune complexes circulating freely in the body fluid and composed ofBDV antigens and specific antibodies attached thereto are detected in abody fluid specimen to be assayed by means of a suitable immunologicalassay.

In the following text, a few further examples are described withreference to figures.

FIGS. 1, 2, 4 and 5 show schematically how the state of the infection(in particular during the disease phase) changes constantly, and that,diagnostically speaking, the measurement of several parameters isoptimal for this type of persistent viral infection (with lowreplication rate during the activation phases). The data plotted are ineach case the absorbance at 405 nm vs. “weeks” (course of the study).The figures show the course for

CICs—circulating immune complexes

cAg—cellular antigen

pAg—plasma antigen

Ab—antibodies (EIA)

FIG. 1 shows the data of human patient 1 (initials D. R.), a 59-year-oldrMDD-patient during hospitalization (treatment with antigen depressants,no antiviral treatment). The examination period was approximately 3months (18 weeks). Assay system: EIA; dilution: cAG 1:2, CIC 1:20, pAG1:2, Ab 1:100; cut-off limit 0.1 specimen origin: for cAG: for cAGsonified PBMCs, otherwise suitable plasma specimens.

FIG. 2 shows a diagram for a 30-year-old OCD patient (OCD=obsessivecompulsive disorder) during hospitalization, the diagram correspondingto FIG. 1. BDV infection plays a not inconsiderable role in thesepatients, like in the case of depressive patients. In both patientgroups—viz. depressive and OCD patients—it is possible thatserotonin-controlled processes are dysfunctional. The reason for thismight be that the BDV virus functionally attacks certain loci in thebrain and on the ganglia. FIGS. 1 and 2 show parameter developmentswhich are similar to each other.

FIG. 3 shows 2 examples from the animal sector, viz. two horsessuffering from Borna disease. In these cases, only one specimen wasexamined. The ratio of CIC to pAG (free plasma antigen) and Ab (=freeplasma antibody (Ab), measured by EIA) can be seen clearly. Specimen4311 was obtained from a horse from Northern Germany (initial G.) whichhad been examined in March 1997 owing to the (questionable) clinicaldiagnosis “Borna disease. The IF antibody titer was 1:40. Specimen 5231was obtained from a horse from Southern Germany (initial D.) which hadbeen examined in May 1997 owing to the clinical diagnosis Borna disease.The IF antibody assay was negative. The horse had fallen ill two monthsearlier, in March 1997, and showed apathy and somnolence. The symptomsthat remained in May were head shaking and stiff gait (EIA parameters:dilution cAG 1:2, CIC 1:20, pAG 1:2, Ab 1:100, cut-off limit=0.1, basefor the specimens: cAg: sonified PBMCs, otherwise suitable plasmaspecimens).

The data on which the figures are based are shown in tabulated formhereinbelow:

TABLE 1 (for FIG. 1): Absorbance (405 nm) at cAG CIC pAG Ab   0 W 0.140.982 0.237 0.051 3 0.041 1.019 0.225 0.177 4 0.051 0.858 0.251 0.146 50.089 0.241 0.237 0.048 6 0.096 0.120 0.299 0.104 7 0.088 0.323 0.2090.163 8 0.081 0.503 0.185 0.158 9 0.056 0.582 0.192 0.11 10  0.068 1.2370.18 0.125 11  0.075 0.91 0.217 0.148 13  0.079 0.366 0.208 0.097 14 0.178 0.79 0.35 0.041 15  0.041 1.179 0.353 0.126 18  0.028 0.397 0.2540.108

TABLE 2 (for FIG. 2): Absorbance (405 nm) at cAG CIC pAG Ab   0 W 0.1080.869 1.335 1 0.117 0.639 0.478 0.367 4 0.146 1.679 0.438 0.28 6 0.2080.421 0.401 0.263 8 0.08 0.675 0.439 0.386 10  0.105 1.174 0.307 0.29612  0.096 14  1.009 0.542 0.307 18  0.134 1.048 0.474 0.417

TABLE 3 (for FIG. 3): Specimen 4311 Specimen 5231 (absorbance)(absorbance) cAG 0.06 0.131 CIC 1.24 0.657 pAG 0.892 1.092 Ab 0.74 0.823

Finally, FIGS. 4a) to d) shows various results for CSF specimens invarious dilutions. FIGS. a) to c) show a high level of positive antigenmarkers in affective disorders and d) shows negative assay results inschizophrenics.

The high values suggest a temporarily copious virus replication in thebrain during the acute depression.

The individual figures show:

Figure a: CSF specimen from patient 36—major depressive disorder, secondepisode, female, 58 years

Figure b: CSF specimen patient 137—major depressive disorder, secondepisode, female, 29 years

Figure d: CSF specimens patient 27: schizophrenia, paranoid type,chronic, male, 39 years; patient 62: schizophrenia, paranoid type,unspecific, female, 37 years; patient 130: schizophrenia, paranoid type,subschronic, female, 20 years

X. Summary

Borna disease virus (BDV) is considered to be the prototype of a newfamily of coated negative-strand RNA viruses. BDV infects nerve cells,but also non-neuronal cells in the brain and the body without destroyingthe cells. BDV infections persist. They are found in humans and a largenumber of domestic animals and livestock. Human BDV infections arehighly likely to play a role in periodic emotional disorders; inanimals, BDV causes periodic behavioral disorders. Individual riskfactors decide on the frequency at which the latent infection isactivated and thus on the disease risk. Symptom-free carriers exist inall species. The diagnosis is made difficult by low viral replicationrates and long latent phases.

The invention allows Borna disease virus (BDV) infections in humans andanimals to be identified virtually completely by combining anewly-discovered assay parameter, CICs in the blood plasma, withparameters which are already known, viz. AG in leukocytes or bloodplasma and Ab in the plasma. All three parameters can be determined on asingle blood specimen (10 ml citrated blood).

The detection, described for the first time in the invention, ofBDV-specific CICs as the only long-term persisting infection markers,allows not only healthy carriers (latent infections) to be identified,but also the course of the infection and the success of the therapy tobe assessed in the case of diseased individuals.

What is claimed is:
 1. A process for detecting Borna disease virus (BDV)infection in an animal, comprising (a) contacting a body fluid specimenwith a first antibody specific for a circulating immune complex (CIC)indicative of BDV infection, which immune complex comprises a BDVantigen and an antibody to BDV antigen, and (b) detecting bindingbetween said first antibody and said immune complex, wherein saidbinding is indicative of infection.
 2. A process according to claim 1,additionally comprising detecting the presence of at least one BDVantigen in said body fluid specimen, by using a second antibodyoriginating from a species different from said first antibody, whereinsaid second antibody is BDV-specific.
 3. A process according to claim 2,wherein said BDV antigen is BDV nucleoprotein p40 or BDV-phosphoproteinp24.
 4. A process according to claim 1, additionally comprisingdetecting the presence of at least one BDV antibody in said body fluidspecimen, by binding a standardized solution of native BDV antigens,prepared from infected tissue culture or from brain of infected animals,to said first antibody, wherein said at least one BDV antibody isindicative of BDV infection and can be detected in said body fluidspecimen.
 5. A process according to claim 2, additionally comprisingdetecting the presence of at least one BDV antibody in said body fluidspecimen, by binding a standardized solution of native BDV antigens,prepared from infected tissue culture or from brain of infected animals,to said first antibody, wherein said at least one BDV antibody isindicative of BDV infection and can be detected in said body fluidspecimen.
 6. A process according to claim 1, wherein said body fluidspecimen is a blood, urine or spinal fluid specimen.
 7. A processaccording to claim 2, wherein the body fluid specimen is a blood plasma,urine or spinal fluid specimen.
 8. A process according to claim 2,wherein the body fluid specimen is a blood specimen, and wherein theantigen detection is carried out on a leukocyte fraction or on a bloodplasma fraction of said blood specimen.
 9. A process according to claim7, wherein all tests are carried out on a blood plasma specimen.
 10. Aprocess for detecting a BVD circulating immune complex (CIC) of anantigen and an antibody which circulates in a body fluid, comprising:(1) contacting a specimen of said body fluid with a support, whereinsaid support has monoclonal or polyclonal antibodies that bind to anantigen in said CIC, said monoclonal or polyclonal antibodies beingfixed to said support via the Fc region wherein said monoclonalantibodies are monoclonal BDV-specific antibodies that are directedagainst native BDV antigens, and wherein said native BDV antigens arederived from natural sources; (2) contacting said specimen from (1) witha secondary antibody of a species other than the assayed species,wherein said secondary antibody is specific for antibodies of thespecies whose body fluid specimen was used; and (3) detecting binding ofsaid secondary antibody to said support.
 11. A process according toclaim 10, wherein the BDV-specific antibodies are selected from thegroup consisting of N protein-specific antibodies and P protein-specificantibodies.
 12. A process according to claim 10, wherein the support isan adsorptively fixing polymer assay plate, which is first occupied ascompletely as possible with secondary antibodies which are specific forthe species from which the immune complex-antigen-specific antibodieswere obtained, and subsequently the immune complex-antigen-specificantibodies are applied to this layer of secondary antibodies.
 13. Aprocess according to claim 10, wherein detection of the secondaryantibody in accordance with (3) of the process is done via an EIA or RIAprocess.
 14. A process according to claim 13, wherein the secondaryantibody is coupled to alkaline phosphatase and is visualized withp-nitrophenyl phosphate by means of a color reaction or made selectableby means of optical detectors.
 15. A diagnostic kit for detecting BDVinfection, comprising at least one BDV-specific monoclonal or polyclonalantibody, means for contacting these antibodies with a specimensuspected of containing BDV antigens or BDV immune complexes, and meansfor detecting the attached antigens or immune complexes.
 16. Adiagnostic kit for detecting BDV infection, comprising at least oneBDV-specific monoclonal or polyclonal antibody occupied by a BDVantigen, means for contacting the antigen-occupied antibodies with aspecimen suspected of containing BDV antibodies, and means for detectingthe attached antibodies.
 17. A diagnostic kit according to claim 15,comprising a unit on or in which the BDV-specific antibodies are presentin immobilized form.
 18. A diagnostic kit according to claim 17, whereinthe BDV-specific antibodies are monoclonal or polyclonal antibodiesobtained from a first species which are immobilized on a support coatedwith a species II-anti-species I IgG obtained from a second, differentspecies.
 19. A diagnostic kit according to claim 18, wherein the supportis a solid plate or an assay tube.
 20. A diagnostic kit according toclaim 18, wherein the antibodies from the first species are polyclonalor monoclonal mouse antibodies.
 21. A diagnostic kit according to claim20, wherein the antibodies from the first species are selected from thegroup consisting of P-protein and N-protein specific monoclonal mouseantibodies, and the adsorptive coating of the support is composed of ananti-mouse IgG.
 22. A diagnostic kit according to claim 21, wherein theanti-mouse IgG is a goat-anti-mouse IgG.
 23. A diagnostic kit accordingto claim 18, wherein the BDV-specific antibodies are immobilized viapolystyrene-bound Clq.
 24. The process of claim 1, wherein said animalis human.
 25. The process of claim 24, wherein said human is sufferingfrom a neurological condition.
 26. The process of claim 25, wherein saidneurological condition is selected from the group consisting ofdepression, and obsessive-compulsive disorder.